O papel das micorrizas arbusculares (MAs) no metabolismo de nitrogênio nos vegetais é pouco conhecido. Pouco se sabe também sobre os mecanismos que regulam a absorção, assimilação de nitrogênio em MAs. Recentemente, um gene altamente induzido em raízes de tabaco micorrizadas foi clonado e caracterizado. Esse gene codifica uma proteína acessória da urease (UreG). Sua regulação diferencial em MAs sugere que fungos micorrízicos arbusculares podem induzir alterações no metabolismo de nitrogênio nos vegetais, e/ou que ureases podem ser importantes no controle do desenvolvimento da simbiose. O objetivo deste trabalho foi verificar se ocorrem ou não alterações nas atividades de ureases em raízes de tabaco micorrizadas, e se essas alterações estão relacionadas com alterações do perfil de aminoácidos solúveis e com o crescimento fingico intrarradicular. Para isso, plântulas de tabaco foram inoculadas com Glomus clarum ou Glomus intraradices, e cultivadas em substrato esterilizado contendo 50, 100 ou 150 mg kg-1 de Nitrogênio, na forma de uréia ou sulfato de amônio. Oito semanas após a inoculação, foram avaliados: matéria seca da parte aérea e sistema radicular, colonização micorrízica intraradicular, atividades de ureases nas raízes, e concentração e perfil de aminoácidos solúveis na parte aérea. A colonização intraradicular por G. clarum teve aumento induzido pelo aumento das doses de nitrogênio. As atividades de ureases de plantas não-inoculadas aumentaram com o aumento da dose de N-uréia aplicada ao substrato. Quando cultivadas com 50 mg kg-1 de N-sulfato de amônio, as raízes de plantas inoculadas com G. clarum apresentaram atividades de ureases mais elevadas do que nos controles não-inoculados. Já, as raízes de plantas inoculadas com G. intraradices apresentaram maiores atividades de ureases, em relação aos controles não-inoculados, quando cultivadas com 100 mg kg-1 de N-sulfato de amônio. Com 150 mg kg-1 N-uréia, as atividades de ureases foram 3 vezes menores nas raízes micorrizadas do que nas não-micorrizadas. As atividades de ureases apresentaram correlação positiva e significativa com a colonização intrarradicular nos tratamentos com N-sulfato de amônio. A inoculação de plântulas com G. intraradices ou G. clarum resultou em diminuição da concentração de aminoácidos livres, em substrato com 50 mg kg-1 N-sulfato de amônio, em relação aos controles não inoculados. Em substrato 150 mg kg-1 N-uréia a concentração de aminoácidos solúveis aumentou aproximadamente 10 vezes, em relação aos controles não-inoculados. O aminoácido encontrado em maior quantidade na parte aérea de tabaco foi a glutamina. Alterações nos perfis de aminoácidos na parte aérea de plantas micorrizadas, em relação aos controles não micorrizados, foram observadas. Correlação significativa entre atividades de ureases nas raízes e concentração de aminoácidos solúveis na parte aérea não foi observada. Os dados obtidos neste trabalho sugerem que as atividades de ureases nas raízes, em condições de baixo N, podem estar relacionadas com a capacidade de colonização intrarradicular dos fungos, e que fungos micorrízicos arbusculares podem alterar o metabolismo de N nas plantas / The role of arbuscular mycorrhizae (AM) in the nitrogen (N) metabolism of host plants is not understood, and the mechanisms regulating N uptake and assimilation in AM are not known. Recently, a gene highly induced in mycorrhizal tobacco roots was cloned and characterized. Its deduced amino acid sequence showed a high degree of homology to urease accessory proteins (UreG). The differential regulation of ureG in AM suggests that arbuscular mycorrhizal fungi may alter N metabolism in the host plants, and/or that ureases may be important in controlling symbiosis development. The aim of this work was to determine whether urease activities are altered in mycorrhizal roots, and whether these alterations are related to changes in the relative abundance of free amino acids in shoots, and/or intraradical fungaI growth. Tobacco seedlings were inoculated with Glomus clarum or Glomus intraradices, and grown in sterilized substrate containing 50, 100 or 150 mg N kg-1, as urea or ammonium sulphate. Eight weeks after inoculation, the following parameters were evaIuated: shoot and root dry weight, intraradical fungaI growth, urease activities in roots, and free amino acid concentration in shoots. IntraradicaI colonization by G. clarum was higher than by G. intraradices, at the different experimental conditions. Root coIonization by G. clarum and G. intraradices was induced in substrate with 50 and 100 mg N-urea kg-1. Urease activities in non-mycorrhizaI roots were higher at the highest N-urea concentration. When grown in 50 mg N-urea kg-1, urease activities in roots coIonized by G. clarum were higher than in non-mycorrhizaI controIs. In contrast, roots coIonized by G. intraradices showed higher urease activities than non-mycorrhizaI controIs when grown in 100 mg N-ammonium sulphate kg-1. Urease activities in mycorrhiza I roots grown in substrate containing 150 mg N-urea kg-1 were 3-fold Iower than in non-mycorrhizal controIs. A significant positive correlation between urease activities and intraradical fungaI growth was observed in roots grown with N-ammonium suIphate. InocuIation of tobacco seedlings with G. intraradices or G. clarum resuIted in decreased free amino acid concentration in the shoots, when grown with 50 mg N-ammonium suIphate kg-1, as compared to the not- inoculated controls. In mycorrhizaI roots grown in substrate with 150 mg N-urea kg<sup-1, free amino acid concentration was approximateIy 10-fold higher than in non- mycorrhizaI controIs. Glutamine was the most abundant amino acid in tobacco shoots. Changes in the reIative abundance of free amino acids in the shoots of mycorrhizal plants, as compared to non-mycorrhizaI controIs, were observed. The data suggest that urease activities in roots, at Iow N concentration, might be involved in the controI of intraradicaI fungaI growth, and that arbuscular mycorrhizaI fungi may induce changes in the amino acid metaboIism in tobacco

Submitted by PPG Medicina e Ci?ncias da Sa?de (medicina-pg@pucrs.br) on 2018-09-17T18:15:32Z No. of bitstreams: 1 KARINE_KAPPAUN.pdf: 4206975 bytes, checksum: b450fa28646271e7dfac9389de975825 (MD5) / Approved for entry into archive by Sheila Dias (sheila.dias@pucrs.br) on 2018-09-18T12:20:56Z (GMT) No. of bitstreams: 1 KARINE_KAPPAUN.pdf: 4206975 bytes, checksum: b450fa28646271e7dfac9389de975825 (MD5) / Made available in DSpace on 2018-09-18T12:54:15Z (GMT). No. of bitstreams: 1 KARINE_KAPPAUN.pdf: 4206975 bytes, checksum: b450fa28646271e7dfac9389de975825 (MD5) Previous issue date: 2018-07-06 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES / Ureases (urea amidohydrolases, EC 3.5.1.5) are nickel-dependent enzymes, widely spread in bacteria, fungi and plants, which catalyze the hydrolysis of urea to ammonia and carbon dioxide. Jaburetox is a peptide derived from a urease isoform of the Canavalia ensiformis plant. Structurally, jaburetox is an intrinsically disordered peptide, demonstrated by bioinformatics tools, and experimentally, by circular dichroism (CD) and nuclear magnetic resonance (NMR). Several biological properties of jaburetox have already been described, such as insecticidal activity, ability to interact with lipid bilayers, fungitoxic effect, among others. Soyuretox is a peptide colinear to jaburetox derived from the soybean ubiquitous urease. One of the objectives of this thesis was to carry out structural studies of the soyuretox peptide by CD and NMR, which revealed its intrinsically disordered nature and that its secondary structure content is modified in the presence of SDS micelles. Biological properties of soyuretox were evaluated. The peptide has entomotoxic activity, inducing aggregation of hemocytes from Rhodnius prolixus, the vector of Chagas disease, both in vivo and in vitro. The fungitoxic effect of soyuretox on yeast Candida albicans involves the production of superoxide anions, and the peptide was immunolocalized on the surface of the yeast. Finally, the behavioral and morphological effects of soyuretox on zebrafish larvae (Danio rerio) were evaluated, aiming to deepen the understanding of the mechanism of toxic action and the toxicological profile of this molecule, which presents biotechnological potential as a biopesticide. / Ureases (ureia amido-hidrolases; EC 3.5.1.5) s?o enzimas n?quel dependentes, amplamente distribu?das em bact?rias, fungos e plantas, que catalisam a hidr?lise da ureia ? am?nia e di?xido de carbono. O jaburetox ? um pept?deo derivado de uma isoforma de urease da planta Canavalia ensiformis. Estruturalmente, o jaburetox ? um pept?deo intrinsecamente desordenado, demonstrado por ferramentas in silico de bioinform?tica, e experimentalmente, por dicro?smo circular (CD) e resson?ncia magn?tica nuclear (RMN). Diversas propriedades biol?gicas do jaburetox j? foram descritas, tais como atividade inseticida, capacidade de interagir com bicamadas lip?dicas, efeito fungit?xico, dentre outras. O soyuretox ? um pept?deo colinear ao jaburetox, por?m derivado da urease ub?qua de soja. Um dos objetivos dessa tese foi realizar estudos estruturais do pept?deo soyuretox por CD e RMN, que revelaram sua natureza intrinsecamente desordenada e aumento no conte?do de estrutura secund?ria na presen?a de micelas de SDS. Propriedades biol?gicas do soyuretox foram avaliadas. O pept?deo tem atividade entomot?xica, induzindo agrega??o de hem?citos de Rhodnius prolixus, o vetor da doen?a de Chagas, tanto in vivo como in vitro. O efeito fungit?xico do soyuretox na levedura Candida albicans envolve a produ??o de ?nions super?xido, sendo o pept?deo imunolocalizado ligado na superf?cie da levedura. Por fim, foram avaliados os efeitos comportamentais e morfol?gicos do soyuretox em larvas de zebrafish (Danio rerio), visando aprofundar o entendimento do mecanismo de a??o t?xica e o perfil toxicol?gico dessa mol?cula, que apresenta potencial biotecnol?gico como biopesticida.

The thesis is divided in to five chapters. Chapter I: Synthesis of New thiolevomannosan derivatives In this chapter, a general and efficient method for the synthesis of conformationally locked thiosugars has been discussed. An unprecedented synthesis of a novel thioorthoester and its synthetic utility in glycosylation has been demonstrated. Chapter II: Studies on -Mannosidase Inhibitors The synthesis and evaluation of novel, conformationally locked glycomimic, thiolevomannosan and its analogs sulfoxide and sulfone starting from readily available D-mannose is discussed in this chapter. This is the first report of thiosugar derivatives with enhanced potency compared to kifunensine. Docking and biochemical studies have been discussed. Chapter III: Studies on Novel Cyclic Tetraselenides of Mannose In this chapter, the syntheses and structural properties of novel cyclic tetraselenides starting from mannose have been discussed. These tetraselenides are the first of their kind where all four selenium atoms are arranged in a cyclic manner as the backbone of mannose. X-ray structures have been reported for the tetraselenides. Chapter IV: Novel Chalcogenides of Uridine and Thymidine: Synthesis and Applications An efficient and simple method to synthesise disulfides and diselenides of thymidine and uridine derivatives has been demonstrated in this chapter. The utility of these disulfides in various ring opening reactions as well in Michael addition reactions has been demonstrated. Chapter V: Studies on New, Potent Urease Inhibitors In this chapter, a facile one-pot synthesis of thio and selenourea derivatives under mild conditions by the reaction of amines and commercially available Viehe’s iminium salt in the presence of benzyltriethylammonium tetrathiomolybdate as sulfur transfer reagent and tetraethylammonium tetraselenotungstate as selenium transfer reagent has been discussed. A few of the urea derivatives have shown potent inhibitor activity in the nanomolar range for jackbean urease.